Protein lysates obtained from EVs isolated by UCF, scUCF, Vn96, ME buffer and Scr peptide (from Fig. 1 a, Step 6ii) were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using the BioRad mini-gel system for canonical EV protein markers as well as Calnexin, an endoplasmic reticulum protein that is not localized in EVs. All antibodies were supplied by Santa Cruz Biotechnology (Dallas, TX) except for antibodies to Flotillin-1 (Cell Signaling Technologies; cat # 18634S) and Calnexin (Abcam, cat # 22,595). Santa Cruz antibodies included CD63 (sc-5275), CD9 (sc-59140), and HSC70 (sc-7298). All primary antibodies were incubated overnight at 4 °C in 5% milk/PBS at a dilution of 1:1,000, with the exception of HSC70 which was used at a dilution of 1:500. Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch Inc. and used at 1:10,000. Clarity Western ECL blotting substrate (Bio-Rad, Hercules, CA) was used for Western blot detection.
Goat anti rabbit and goat anti mouse secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Goat anti-rabbit and goat anti-mouse secondary antibodies are used to detect the presence of primary antibodies raised in rabbits or mice. These secondary antibodies are labeled with enzymes or fluorescent dyes, allowing visualization and quantification of the target proteins or molecules in various immunoassays and techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Mini gel system, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Flotillin 1, by Cell Signaling Technology (1 mentions)
Calnexin, by Abcam (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Nf κb p65, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ecl advance detection kit, by GE Healthcare (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
5 protocols using goat anti rabbit and goat anti mouse secondary antibodies
1
EV Protein Characterization by Western Blot
2
Protein Extraction and Western Blot Analysis
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Total proteins in MG63 cells were extracted with loading buffer (2×) and the concentrations were determined with a BCA protein assay kit (Beyotime Biotechnology Company, Shanghai, China) according to the specifications. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% separating gel and 5% stacking gel. Then, the protein was transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore Corporation, Darmstadt, Germany), and the membranes were blocked with 5% non-fat milk and 2% bovine serum albumin (BSA, Gen-View Scientific Inc., USA) in PBS-T (PBS containing 0.25% Tween-20, Macklin) at 25°C for 1.5 h. The membranes were incubated with rabbit anti-IGF-1R primary antibodies (1: 4000, Proteintech Group Inc., Wuhan, China) and mouse anti-β-actin (1: 10 000, Proteintech), as internal control, at 4°C overnight. The next day, the membranes were incubated with goat anti-rabbit and goat anti-mouse secondary antibodies (1: 3000, Jackson ImmunoResearch Laboratories, Inc. USA) at 25°C for 2 h, and then analyzed with electrochemiluminescence (ECL, Millipore, MA, USA).
3
Western Blot Analysis of EMT and NF-κB Markers
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Proteins were extracted from cell lysate with nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, Shanghai, China) and then quantified with BCA protein assay kit (Beyotime). Proteins were analyzed by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and 5% stocking gel, and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Corporation, Shanghai, China). Protein bands were incubated with E-cadherin (1: 1000, Proteintech Group, Inc. Wuhan, China), vimentin (1: 1000, Proteintech), Ezrin (1: 1000, Proteintech), p-Ezrin Tyr353 (1: 1000, Abcam, Shanghai, China), p-Ezrin Thr567 (1: 1000, Abcam), NF-κB p65 (1: 3000, Abcam), p-IκBα Ser32/36 (1: 1000, CST), and GAPDH (1: 10000, Proteintech) primary antibodies at 4°C overnight, and then incubated with goat anti-rabbit and goat anti-mouse secondary antibodies (1: 3000, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) at 25°C for 1 h. An electrochemiluminescence (ECL, Millipore) system was used to expose proteins.
4
Protein Extraction and Western Blot Analysis
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The proteins are extracted from the different cell lines with RIPA buffer (Merck-Millipore, Bruxelles, Belgium), 1× complete protease inhibitor cocktail (Roche, Mannheim, Deutschland), and phosphatase inhibitors (Sigma, Overijse, Belgium). The protein concentrations were determined by Bradford assay (BioRad, Hemel Hempstead Herts HPS2 7DK, UK). Thirty micrograms of protein samples were separated on 12% SDS-polyacrylamide gels, then transferred onto PVDF (Polyvinylidene difluoride) membrane for 1.5 h. The membrane are blocked 3 h with 5% milk of solution in TBST (Tris-buffered saline/tween), and incubated overnight at 4 °C with the primary antibodies. Goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson Immuno Research Laboratories, Sulfolk, UK) were used as appropriate, and detected with the ECL Advance Detection Kit (GE Healthcare, Diegen, Belgium) on Kodak films (Thermofisher, Merelbeke, Belgium).
5
Liraglutide Modulates FoxO1-Mediated Autophagy in Insulinoma Cells
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INS-1 rat insulinoma cells were purchased from the American Type Culture Collection. RPMI-1640 medium was purchased from Thermo Fisher Scientific, Inc. FBS was purchased from Hangzhou Sijiqing Biological Engineering Materials Co. Ltd. Liraglutide was purchased from Novo Nordisk Hellas Ltd. PA and chloroquine (CQ) were obtained from Sigma-Aldrich (Merck KGaA). FoxO1 small interfering RNA (siRNA) was obtained from Shanghai GenePharma Co., Ltd. The following antibodies were used at a 1:1,000 dilution: Microtubule-associated protein 1 light chain3 (LC3; cat. no. 2775; Cell Signaling Technology, Inc.), phosphorylated (p)-FoxO1 (cat. no. 9461; Cell Signaling Technology, Inc.), FoxO1 (cat. no. 2880; Cell Signaling Technology, Inc.), cleaved caspase-3 (cat. no. AF1150; Beyotime Institute of Biotechnology), β-actin (cat. no. 8457; Cell Signaling Technology, Inc.) and GAPDH (cat. no. sc-32233; Santa Cruz Biotechnology, Inc.). Horseradish peroxidase-conjugated secondary antibodies were used at a 1:5,000 dilution, which included goat anti-rabbit and goat anti-mouse secondary antibodies from Jackson ImmunoResearch Laboratories, Inc. (cat. nos. 111-545-003 and 115-005-003). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. SDS-PAGE and an ECL detection kit were obtained from Cytiva.
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